Exosomes are secreted nanovesicles which incorporate proteins and nucleic acids, thereby enabling multifunctional pathways for intercellular communication. There is an increasing appreciation of the critical role they play in fundamental processes such as development, wound healing and disease progression, yet because of their heterogeneous molecular content and low concentrations in vivo, their detection and characterization remains a challenge. In this work we combine nano- and microfabrication techniques for the creation of nanosensing arrays tailored toward single exosome detection. Elliptically–shaped nanoplasmonic sensors are fabricated to accommodate at most one exosome and individually imaged in real time, enabling the label-free recording of digital responses in a highly multiplexed geometry. This approach results in a three orders of magnitude sensitivity improvement over previously reported real-time, multiplexed platforms. Each nanosensor is elevated atop a quartz nanopillar, minimizing unwanted nonspecific substrate binding contributions. The approach is validated with the detection of exosomes secreted by MCF7 breast adenocarcinoma cells. We demonstrate the increasingly digital and stochastic nature of the response as the number of subsampled nanosensors is reduced from four hundred to one.

Severe acute respiratory syndrome-associated coronavirus 2 is a major global health issue and is driving the need for new therapeutics. The surface spike protein, which plays a central role in virus infection, is currently the target for vaccines and neutralizing treatments. The emergence of novel variants with multiple mutations in the spike protein may reduce the effectiveness of neutralizing antibodies by altering the binding activity of the protein with angiotensin-converting enzyme 2 (ACE2). To understand the impact of spike protein mutations on the binding interactions required for virus infection and the effectiveness of neutralizing monoclonal antibody (mAb) therapies, the binding activities of the original spike protein receptor binding domain (RBD) sequence and the reported spike protein variants were investigated using surface plasmon resonance (SPR). In addition, the interactions of the ACE2 receptor, an anti-spike monoclonal antibody (mAb1), a neutralizing monoclonal antibody (mAb2), the original spike RBD sequence, and mutants D614G, N501Y, N439K, Y453F, and E484K were assessed. Compared to the original RBD, the Y453F and N501Y mutants displayed a significant increase in ACE2 binding affinity, whereas D614G had a substantial reduction in binding affinity. All mAb-RBD mutant proteins displayed a reduction in binding affinities relative to the original RBD, except for the E484K-mAb1 interaction. The potential neutralizing capability of mAb1 and mAb2 was investigated. Accordingly, mAb1 failed to inhibit the ACE2-RBD interaction and mAb2 inhibited the ACE2-RBD interactions for all RBD mutants, except mutant E484K, which only displayed partial blocking.

Carcinoembryonic antigen (CEA), also referred as CEACAM5, is integral to the adhesion process during cancer invasion and metastasis and is one of the most widely used tumor markers for assisting the diagnosis of cancer recurrence and cancer metastasis. Antibodies against CEA molecules have been developed for detection and diagnostic applications following tumor removal. Single domain antibodies (sdAbs) against CEA isolated from dromedary and llama exhibited high specificity in binding to tumor cells. However, because these CEA sdAbs were not designed to be orientated when conjugated to surface sensors, there is potential for significant improvements in their activity and limit of detection. Herein we modified the CEA sdAbs with two different C-terminal fusions designed to aid with orientation by way of the tail’s charge and biotin binding. A fusion which incorporated the C-terminus addition of a positively charged tail (B5-GS3K) improved biosensor sensitivity to CEA while also retaining the sub-nanomolar binding affinity and thermal stability of the unmodified sdAb. Using our fabricated surfaces on bare gold chips and a multiplexed surface plasmon resonance imager (SPRi), we quantified the specific binding activities, defined as the percentage of bound epitopes to the total immobilized, of the sdAb fusions and anti-CEA mAb. Our results demonstrate that monovalent B5-GS3K exhibited significantly improved binding activity, approximately 3-fold higher than bivalent mAb.

Abstract

A fabrication method for high-throughput, fiber-based tips for near-field scanning microscopy (NSOM) in the mid-infrared (λ ~ 3 μm) has been developed. Several fiber materials have been investigated and recipes for wet-chemical etching have been varied to produce tips that are physically robust and are capable of low-loss transmission of high-power pulses of mid-infrared light. Ultimately, wet-chemical etching techniques are used on glass fibers to produce tips capable of focusing mid-infrared light to ablate material from sub-micron-sized regions of organic films. The power throughput of the tips is significantly increased by using a novel material, previously unreported for NSOM applications: germanate fibers. The tips produced are mechanically strong and capable of transmitting high light fluence without sustaining physical damage. Here, the development of these tips and their performance are described.